Function of O-fucose glycans in Notch signaling

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Abstract

Notch receptors are single transmembrane glycoproteins containing &sim;29-36 epidermal growth factor-like (EGF) repeats in the extracellular domain. Many EGF repeats are modified with O-fucose by protein O-fucosyltransferase-1 (Pofut1). The O-fucose may be extended with N-acetylglucosamine, galactose and sialic acid and is required for Notch receptors to respond optimally to signaling from canonical Notch ligands. Inactivation of mouse Pofut1 leads to embryonic lethality at &sim;E9.5 and global Notch signaling defects. The first aim of this thesis was to identify roles for Notch signaling during spermatogenesis in the mouse using conditional deletion of Pofut1 in primary spermatocytes, spermatids and Sertoi cells using Scp1-Cre, Protamine1-Cre and AMH-Cre recombinase transgenes, respectively. Pofut1F/F males carrying each of these trangenes were fertile, and had normal litter sizes. There were no detectable morphologic changes in adult testes and no apparent increase in apoptotic cells. My data suggest that the deletion efficiency of Pofut1 by these Cre transgenes was low, and that partial deletion of Pofut1 did not affect spermatogenesis.;The second aim of this thesis was to investigate the role of a putative GDP-fucose transporter Slc35c2 in Notch signaling. I showed that fibroblasts from mice or humans lacking the well-established Golgi GDP-fucose transporter Slc35c1 exhibited more robust Notch signaling than controls in Notch co-culture signaling assays. The O-fucosylation of a Notch1 EGF repeat fragment was increased in CHO transfectants overexpressing Slc35c2. In CHO cells with low levels of Slc35c2, both Delta1- and Jagged1-induced Notch signaling were reduced, and the O-fucosylation of a Notch1 fragment was also decreased. Immunofluorescence microscopy and cell fractionation experiments showed that Slc35c2 is primarily colocalized with markers of the cis Golgi network and ERGIC. The combined results suggest that Slc35c2 enhances the O-fucosylation of Notch and is required for optimal Notch signaling in mammalian cells. Mice lacking Slc35c2 were viable and fertile and had no gross developmental Notch signaling defects. Double mutants lacking both Slc35c1 and Slc35c2 were born and preliminary data indicate that T-cell development, which depends on Notch signaling, may be unaffected in Slc35c2 null mutants. The combined data implicate another transporter in providing GDP-fucose for the fucosylation of Notch receptors.